Novel type III secretion pathway in Aeromonas salmonicida, and uses therefor

ABSTRACT

Disclosed is a newly identified and characterized type III secretion system in  Aeromonas salmonicida . The invention also encompasses the use of components of the novel secretion system in immunoprotection against  A. salmonicida  infection, as well as other diagnostic and therapeutic uses thereof.

FIELD OF THE INVENTION

This invention relates to bacterial secretion systems, and in particularto a newly identified and characterized type III secretion system inAeromonas salmonicida. The invention also encompasses the use ofcomponents of the novel secretion system in immunoprotection against A.salmonicida infection, as well as other diagnostic and therapeutic usesthereof.

BACKGROUND OF THE INVENTION

Various publications are referenced throughout this publication, andfull citations for each of these publications are provided at the end ofthe Detailed Description.

Aeromonas salmonicida, a Gram-negative, facultatively anaerobic,non-motile, rod shaped bacterium, growing at temperatures around 20° C.,is the etiological agent of furunculoses in salmonids, causing mostsevere economic losses in production farms of salmon and trout. Thedisease is characterised in the sub-acute or chronic form by thepresence of haemorrhagic necrotic lesions in the gills, gut and muscle,while in the acute form fish die apparently from toxemia without showingparticular external signs.

Due to the high contagiousity of the disease and the high mortality insalmon of all ages, particularly in the sea water growers, large amountsof antibiotics are used in closed and open waters for therapy offurunculoses (Munro and Hastings, 1993). Vaccination has become animportant strategy to control furunculoses in fish farms (Ellis, 1997).However, the currently applied whole cell antigen vaccines seem to showconsiderable variability in efficacy, the origin of which remainscurrently unexplained (Thornton et al., 1993).

Knowledge of the mechanisms of pathogenicity of A. salmonicida, and inparticular of the main virulence factors involved, is essential in thedevelopment of efficient strategies to prevent outbreaks of furunculosescaused by A. salmonicida. Currently, several potential virulence factorsof A. salmonicida have been reported, including a surface-layer protein(Chu et al., 1991), the hemolysins ASH1, ASH3, ASH4 (Hirono and Aoki,1993), salmolysin (Titball and Munn, 1985), the serine protease AspA(Whitby et al., 1992) and the glycerolipid-cholesterol acyltransferase(GCAT) (Lee and Ellis, 1990), but their role in pathogenesis is unclearand many of them seem not to play a primary role in virulence. This wasdemonstrated by A. salmonicida strains with deletion mutants of the GCATand aspA genes which had no influence on virulence of the strains ininducing furunculoses.

SUMMARY OF THE INVENTION

A new ADP-ribosylating toxin named AexT (Aeromonas exoenzyme T) encodedby the gene aexT was identified in a virulent strain of A. salmonicida.A. salmonicida strains that were propagated for several passages onculture medium had lost expression of AexT, but still retained the aexTgene. AexT shows amino acid sequence similarity to theADP-ribosyltransferase toxins ExoS and ExoT of Pseudomonas aeruginosawhich are secreted by a type III-dependent secretion mechanism (Yahr etal., 1996). Regulation of aexT was shown to be dependent on contact withfish cells and could also be induced by Ca²⁺ depletion of the medium.The aexT gene was found to be preceded by a consensus sequence forbinding of a transcriptional activator known in P. aeruginosa as ExsAwhich is involved in type III mediated gene expression (Frank, 1997).

Based on these observations, we used broad range gene probes to identifyin A. salmonicida a novel type III secretion system by means of the geneacrD (Aeromonas calcium response D) encoding a transmembrane spanningprotein. The acrD gene has a high similarity to IcrD, a protein of theYersinia sp. which is an inner membrane protein of the type IIIsecretion apparatus in Yersinia sp. The acrD gene is flanked by furthertypical type III secretion genes which were designated acr1, acr2, acr3,acr4, acrD, acrR, acrG, acrV, and acrH, and which show significantsimilarity to pcr1, pcr2, pcr3, pcr4, pcrD, pcrR, pcrG, pcrV, and pcrHof Pseudomonas aeruginosa and to tyeA, sycN, yscX, yscY, lcrD, lcrR,lcrG, lcrV, and lcrH of Yersinia enterocolitica. All these genes play apredominant role in building up the type III secretion apparatus in therespective bacterium, including the regulation of the low calciumresponse (LCR) and chaperon functions. The genes isolated from A.salmonicida belong to the analogue of the virA operon, which is centralin the type III secretion pathway of many Gram-negative pathogens ofhuman, animals and plants (Fenselau et al., 1992; Gough et al., 1992;Michiels and Cornelis, 1991).

We have also determined that the type III secretion system in A.salmonicida is located on a 84 kb plasmid which is rapidly lost upongrowth in culture medium. Biosynthesis of AcrV in A. salmonicida, theanalogue to LcrV in Yersinia, requires as a trigger either low Ca²⁺conditions or contact with fish cells. Upon infection with A.salmonicida expressing AcrV, the cultured cells undergo significantmorphological changes. Cultures derived from originally virulent A.salmonicida strains, which had lost the type III secretion genesincluding AcrV, lost virulence as they did not affect rainbow troutgonad cells morphologically after infection. Concomitantly to loss ofthe type III secretion genes, these cultures lost the expression of theaexT gene which specifies the ADP-ribosylating toxin of A. salmonicida.

Rainbow trout gonad cells infected with the virulent A. salmonicida andincubated in antiserum directed against recombinant AcrV-His proteincould be protected from the toxic effect and showed only weakmorphological changes. AcrV, which belongs to the type III secretionproteins is a determinative factor involved in virulence mechanisms ofA. salmonicida, and is expected to provide new insights into basicmechanisms of pathogenicity of bacterial species. The components of thetype III secretion system of A. salmonicida may be used as antigens forthe development of sub-unit vaccines against infection of fish by A.salmonicida.

In one embodiment, the invention comprises an isolated 5.7 kb nucleicacid segment (SEQ ID NO:10) containing the type III secretion genes ofA. salmonicida. In another embodiment, the invention comprises a nucleicacid segment that encodes protein having the amino acid sequence of SEQID NOS:1, 2, 3, 4, 5, 6, 7, 8, and 9, including variants that retaineither biological activity or immunogenicity or both. Due to thedegeneracy of the genetic code and the possible presence of flankingnucleic acid fragments outside of the coding regions, it will beunderstood that many different nucleic acid sequences may encode theamino acid sequence of SEQ ID NO NOS:1, 2, 3, 4, 5, 6, 7, 8, or 9, andvariants, and that all such sequences would be encompassed within thescope of the present invention.

In a further embodiment, the invention relates to the use of AcrV as animmunogen, and to the use of AcrV in a recombinant or traditionalvaccine to reduce the incidence of infection by A. salmonicida.

In another embodiment, the invention provides a means of diagnosing A.salmonicida, or other bacteria found to contain AcrV homologues, by thedetection of the AcrV protein or the homologous proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a genetic map of the type III secretion genes found in A.salmonicida. Boxes with arrowheads indicate open reading frames (ORFs).The size of the different genes (in kilobases) is shown by the scalebar. A restriction map containing restriction enzymes SacI, PstI, NotI,BamHI, and SalI is shown. Abbreviation used: acr, Aeromonas calciumresponse.

FIG. 2 is a segregation curve of A. salmonicida JF2267. An A.salmonicida JF2267 LB-culture was first incubated 2½ hrs at 19° C. andthen at 22° C. for 7 hrs. Colony-blotting was performed to analyze theLB-culture at 10 different time points for positive, respectivelynegative colonies.

FIG. 3 shows a pulsed-field gel electrophoresis of A. salmonicida strainJF2267, and strain JF2397. (Lane 1) JF2267, undigested. (Lane 2) JF2397,undigested. (Lane 3) JF2267 digested with NotI. (Lane 4) JF2397 digestedwith NotI. (Lane 5) Low Range PFG Marker (New England Biolabs). Thewhite arrows indicate the bands that hybridized on Southern blots withthe acrD gene probe.

FIG. 4 shows infection of fish cells with A. salmonicida ATCC 33658^(T),JF2267, and JF2397. RTG-2 cells infected with JF2267 (A), ATCC 33658^(T)(B), JF2397 (C), and pure PBS (D). RTG-2 cells infected with JF2267 andmonospecific polyclonal antibodies against AcrV were protected (E),whereas RTG-2 cells infected with JF2267 and anti-AcrV preserum werenot. Pictures were taken 24 hrs after infection, respectively 21 hrsafter the protection assay under a phase contrast microscope.

FIG. 5 shows low Ca²⁺ response induced AcrV expression in A. salmonicidaJF2267. The picture shows an immunoblot reacted with specific rabbitanti-AcrV antiserum. Strains ATCC 33658^(T) (lane 2), JF2267 (lane 3)and JF2397 (lane 4) were grown in Ca²⁺ depleted medium, harvested bycentrifugation and analyzed on 15% SDS PAGE followed by immunoblotting.Lane 1 contains purified recombinant AcrV-His protein as a control.

DETAILED DESCRIPTION

A 5.7 kb segment containing type III secretion genes of A. salmonicidathat were cloned and sequenced correspond to the pcr locus (Pseudomonascalcium response) of Pseudomonas aeruginosa (Frank, 1997; Yahr et al.,1997b) and the virA operon and genes of the following operon of Yersiniaenterocolitica (Cheng and Schneewind, 2000; Iriarte and Cornelis, 1999;Plano et al., 1991; Skrzypek and Straley, 1993; Motin et al., 1994;Price and Straley, 1989) and other Gram-negative animal and plantpathogens (Fenselau et al., 1992; Gough et al., 1992; Michiels andCornelis, 1991). The most conserved gene at this locus was revealed tobe the acrD gene encoding the AcrD protein, which showed 82% identicalaa to the transmembrane spanning core proteins LcrD of the injectisomeof the Y. enterocolitica type III secretion apparatus and PcrD of theinjectisome of the P. aeruginosa type III secretion apparatus (Yahr etal., 1997b; Plano et al., 1991). Due to this high similarity, weconclude AcrD to have the analogous functions in the injectisome of theA. salmonicida type III secretion pathway.

The least conserved protein encoded on the cloned and analyzed segmentis AcrV, which shows only 35% identical aa to PcrV of P. aeruginosa and37% identity to LcrV of Y. enterocolitica. The main role of LcrV andPcrV, and accordingly also of AcrV, is assumed to be involved in sensingthe bacterium-host interactions (Sawa et al., 1999; Bergman et al.,1991). We therefore interpret the significantly higher dissimilaritybetween AcrV and LcrV or PcrV, compared to the other gene products ofthe type III secretion locus (Table 2), to be due to the hostspecificity which seems to be determined by AcrV, LcrV or PcrV.

Our analyses revealed the A. salmonicida type III secretion genes to belocated on a plasmid of 84 kb. The plasmid was shown to be lost veryeasily in standard growth media, in particular after a slight raise ingrowth temperature. Concomitant to the loss of the type III genes in A.salmonicida, we detected the loss in virulence of the strain as measuredby the infection of RTG-2 fish cell cultures, as well as the loss ofproduction of ADP-ribosylating toxin aexT in supernatants and bacterialcell pellets of low Ca²⁺ response induced A. salmonicida cultures. It isalso noted that AexT biosynthesis induced by contact of A. salmonicidawith RTG-2 fish cells disappeared in those strains or subcultures thathad lost the type III secretion genes. Expression of the aexT gene musttherefore be regulated by a mechanism which is dependent on type IIIsecretion genes. In this context it must be noted that several genes ofthe type III secretion pathway of Yersinia spp., in particular LcrV, aredown regulated and secretion and production of effector proteins iscompletely blocked in the presence of millimolar amounts of Ca²⁺(Forsberg et al., 1987). It also became apparent from tissue cultureinfection models that the absence of Ca²⁺ in vitro mimics a yetundefined signal that is received by Yersinia species when they areadherent to eukaryotic cells and that induce both type III secretiongenes and effector molecules such as YopE and Yops (Cornelis, 1998).

The dependence of aexT expression on type III secretion mechanism wasalso indicated by the presence of a consensus sequence upstream the aexTtoxin gene in A. salmonicida, which shows full homology to the bindingsite of a transcriptional activator, known in P. aeruginosa as ExsA,which is involved in type III dependent gene expression (Frank, 1997).The expression of aexT in A. salmonicida is thus dependent on afunctional type III secretion mechanism. The lack of production of AexTas detected in the type strain of A. salmonicida ATCC 33658^(T) as wellas in the strain JF2397 which was derived from an originally virulent A.salmonicida strain, JF2267, in spite of the presence of a functionalaexT gene, must therefore be due to the loss of the type III secretionpathway.

The AcrV protein of the novel type III secretion pathway of A.salmonicida plays an important role in pathogenesis by its role as asensor and regulator of the system, as shown in other type III secretionsystems. An important role in the secretion-related regulatory role inthe low Ca²⁺ response of Y. pestis is attributed to LcrV, which islocalized to the bacterial surface and required for targeting of Yops ofY. pestis (Fields and Straley, 1999; Nilles et al., 1997). In addition,it was postulated that LcrV is also secreted by a special pathway whichresults its localization in the cytosol of infected cells but not thesurrounding medium (Fields and Straley, 1999). Using a tissue cellmodel, it was shown that antiserum directed against LcrV prevented Y.pestis from injecting the Yop effector molecules into the host cells(Pettersson et al., 1999; Hueck, 1998). Active immunization of mice withrecombinant LcrV antigen efficiently protected mice against challengewith Y. pestis (Leary et al., 1995). Our results showed that antibodiesdirected against recombinant AcrV, the analogous protein to LcrV,protected fish RTG-2 cells from damage caused by virulent A. salmonicidastrain JF2267 and demonstrated that the AcrV plays an important role intype III secretion pathway mediated virulence of A. salmonicida.

The newly found type III secretion pathway plays a central role inpathogenicity of A. salmonicida via the secretion and direct injectionof the ADP-ribosylating toxin AexT into the target cells. Loss of thetype III secretion pathway, which is frequently observed, is due to theinstability of a kb plasmid under culture conditions. Furthermore, lossof type III secretion genes such as acrD and acrV abolished expressionof the aexT gene, and led to loss of virulence of A. salmonicida. Asshown, surface exposed gene products of this type III secretion pathway,in particular AcrV, are potent candidates for new vaccines for theimmune prophylaxis of fish against furunculosis.

The invention is further described by way of the following examples andresults, which are not to be considered as limiting the scope of theinvention. It will be appreciated by those skilled in the art, in lightof this disclosure, that many changes can be made in the specificembodiments disclosed without departing from the scope of the invention.

EXAMPLES AND RESULTS

Materials and Methods

Bacterial Strains, Growth Conditions and Cloning Vectors:

A. salmonicida strains are listed in Table 1. A. salmonicida type strainATCC 33658^(T) was purchased from the American Type Culture Collection.A. salmonicida strain JF2267 was freshly isolated from an arctic char(Savelinus alpinus) showing typical symptoms of furunculoses. A.salmonicida strain JF2397 was derived from strain JF2267 by repeatedsingle colony isolations after each of nine passages propagated on LBagar medium at 22° C. for two days each passage. A. salmonicida strainswere routinely cultured on blood agar plates (Trypticase soy agarsupplemented with 0.1% CaCl₂ and 5% sheep blood) at 19° C. unlessotherwise mentioned.

Liquid cultures of A. salmonicida were made by inoculation of Tripticasesoy broth (TSB) (2.75 g/100 ml Tripticase soy broth without Dextrose(BBL® 11774, Becton Dickinson AG, Basle, Switzerland), 0.1% Glycerol,0.1 M L-Glutamic acid pH 7.3) with fresh culture from solid medium andsubsequent growth for 18 h at 19° C. For growth in Ca²⁺-restrictedmedium, TSB was supplemented with 10 mM Nitrilotriacetic acid (TitriplexI, Merck 1.08416, Darmstadt, Germany).

For cloning and expression of cloned genes, Escherichia coli strains.XL1-blue (recA1 endA1, gyrA96 thi-1 hsdR17supE44 relA1 lac [F′ proABlacI^(q)ZΔM15 Tn10 (Tet^(T))] (Bullock et al., 1987), and BL21 (DE3)(F′dcm ompT hsdS(r_(B)- m_(B)-) gal λ(DE3)) (Studier et al., 1990)respectively, were used. Plasmid pBluescriptII-SK⁻ (Stratagene, LaJolla, Calif., USA) was used as basic cloning vector. For theconstruction of genes encoding poly-Histidine fusion proteins and theirexpression, plasmid pETHIS-1, a T7 promoter based expression vector(Schaller et al., 1999) was used. E. coli strains were grown at 37° C.in Luria-Bertani broth (LB) supplemented when necessary with ampicillin(50 μg/ml) for selection and maintenance of recombinant plasmids. Whenblue-white selection with pBluescriptIISK⁻ was performed, 125 μM X-Galmedium was supplemented with5-bromo-4-chloro-3-indolyl-β-D-thiogalacto-pyranoside.

Preparation of Genomic DNA, Cloning and Sequencing Procedures:

Genomic DNA of A. salmonicida was extracted by the guanidiumhydrochloride method (Pitcher et al., 1989). A partial gene library of,A. salmonicida JF2267 was constructed by cloning agarose gel purifiedSacI-SalI digested fragments of 4 to 6 kb size into vectorpBluescriptII-SK⁻ using standard procedures (Ausubel et al., 1999).Recombinant plasmids were screened by colony blot (Ausubel et al., 1999)using digoxigenin (DIG)-labeled DNA probes as described previously(Braun et al., 1999). Plasmids from A. salmonicida were purified usingthe method of Birnboim and Doly (Birnboim and Doly, 1979).

To construct a genomic library from A. salmonicida JF2267, 0.1 μg of DNApartially digested with Sau3a was ligated to ZapExpress BamHI preparedarms (Pharmacia, Uppsala, Sweden) and packed into phage Lambda.Two-hundred μl of freshly grown XL1-blue MRF′ cells (Pharmacia)resuspended in 10 mM MgSO₄ were infected with the packed phages during15 min at 37° C. Three ml of preheated (50° C.) Top Agarose (LB-brothcontaining 0.7% Agarose) supplemented with IPTG and X-Gal for blue/whiteselection were added and the mixture was poured onto an LB-Agar plate.Plates were incubated overnight at 37° C. and then used for screening ofplaques. Positive plaques were cut out and stored overnight at 4° C. in0.5 ml SM-buffer (100 mM NaCl, 8 mM MgSO₄, 50 mM Tris, pH 7.5, and 0.01%gelatine) containing 20 μl chloroform. 20 ml overnight cultures ofXL1-blue MRF′ grown in LB supplemented with 0.2% maltose and 10 mM MgSO₄and 20 ml XLOLR cells (Pharmacia) grown in LB media were centrifuged for5 min at 4,000 rpm and resuspended in 10 mM MgSO₄ to a final OD₆₀₀=1.Two-hundred μl the XL1-blue MRF′ cells were added to 250 μl of theSM-buffer containing the positive phages and 1 μl (10⁷ pfu) ExAssist™helper phage. This mixture was incubated 15 min at 37° C. and 3 mlLB-broth were added and shaken another 3 hrs at 37° C. The cultures werethen heated for 15 min at 70° C., centrifuged during 15 min at 5,700rpm, 4° C., and the supernatant containing the pBK-CMV phagemidfilamentous phage was decanted into fresh tubes. Two-hundred μl XLOLRcells were mixed with 100 μl supernatant and incubated for 15 min at 37°C., 300 μl LB-broth were added and the culture was incubated for anotherone hr at 37° C. Two-hundred μl of this culture were plated on LB-platescontaining 50 mg/l kanamycin overnight at 37° C. Colonies were pickedand mini-preps (using the QIAprep Spin Miniprep kit, Qiagen AG, Basle,Switzerland) performed for plasmid purification.

For sequencing, subclones of sequential DNA segments were generated witha double-stranded nested deletion kit (Pharmacia LKB, Biotechnology AB,Uppsala, Sweden). Sequencing was done with the dRhodamine TerminatorCycle Sequencing Kit (Applied Biosystems, Foster City, Calif., USA)according to the manufacturer's protocol using either T3 and T7 primersflanking the cloned inserts in pBluescriptII-SK⁻ or customer-synthesizedinternal primers. All sequences were determined on both strands.Reaction products were analyzed on an ABI Prism 310 genetic analyzer(Applied Biosystems).

Sequence Data Analyses:

Sequence alignment and editing were performed by using the softwareSequencher (Gene Codes Corporation, Ann Arbor, Mich., USA). Comparisonsof DNA sequences and their deduced amino acid sequences withEMBL/GenBank and NBRF databases were performed using the programsBLASTN, BLASTX and BLASTP (Altschul et al., 1990). Potentially antigenicsegments of AcrV were determined using the software ProtScale(http://www.expasy.ch/cgi-bin/protscale.pl (Bairoch et al., 1995) andthe software Coils output(htpp://www.ch.embnet.org/software/COILS_form.html) (Lupas et al.,1991). The molecular masses of the protein and its theoreticalisoelectric pH (pI) were calculated by using ProtParam tool(http:/www.expasy.ch/tools/protparam.html) (Gill and von Hippel, 1989).Transmembrane prediction of the protein were made by using Tmpred(http://www.ch.embnet.orgsoftware/TMPRED form.html) (Hofmann andStoffel, 1993).

PCR Amplifications and Preparations of DIG-Labeled Gene Probes:

Template DNA was produced either by extraction of genomic DNA or bypreparation of lysates from bacterial colonies. Lysates were obtained byresuspending five colonies of the corresponding bacterial cultures in200 μl lysis buffer (100 mM Tris-HCl, pH 8.5, 0.05% Tween 20 (Merck),0.24 mg/ml proteinase K (Roche Diagnostics, Rotkreuz, Switzerland)dissolved in pyrogen-free water, filtered through a 0.22 μm low proteinbinding membrane filter) followed by subsequent incubation for 60 min at60° C. and 15 min at 97° C. Lysates were then cooled on ice and used asPCR templates.

PCR amplifications were performed with either a PE9600 or PE2400automated thermocycler with MicroAmp tubes (Applied Biosystems). Thereaction was carried out in a 50 μl reaction mix (10 mM Tris-HCl, pH8.3, 1.5 mM MgCl₂, 50 mM KCl, 0.005% Tween 20, 0.005% NP-40 detergent,170 μM of each deoxinucleoside triphosphate (dATP, dCTP, dGTP, dTTP),0.25 μM of each primer, 2.5 units Taq DNA polymerase (RocheDiagnostics)), and 100 ng of template DNA or 5 μl lysate. For theproduction of DIG-labeled probes, PCR mixtures were supplemented with 40μM digoxigenin-11-dUTP (Roche Diagnostics). PCR conditions were asfollows: 3 min at 94° C. followed by 35 cycles of 30 s at 94° C., 1 minat the corresponding annealing temperature (Table 2), and 30 s at 72° C.In addition, an extension step of 7 min at 72° C. was added at the endof the last cycle in order to ensure fall length synthesis of thefragments.

Curing of Type III Secretion Genes from A. salmonicida:

In order to study the segregation of the type III secretion genes in A.salmonicida strain JF2267, the strain was inoculated in LB-broth at adensity of A₆₀₀=0.08 and incubated 2½ hrs at 19° C. Then the culture wassplit in two. One part was kept for continued growth at 19° C., whilethe other part was incubated at 22° C. Samples were taken at differenttime points from both cultures and spread on LB-agar medium. The plateswere then incubated at 19° C. for 24 hrs. Subsequently, colony blothybridizations were performed using gene probes to determine the loss ofspecific genes.

Pulsed-Field Gel Electrophoresis (PFGE):

The bacterial strains A. salmonicida JF 2267 and JF2397 were grown on LBagar for one day at room temperature. Then bacterial suspensions in 10mM Tris, 10 mM EDTA, pH 8.0, sterile, were prepared to a final OD₆₀₀ of5. Three-hundred μl of 1.5% Sea Kem gold agarose (FMC Bioproducts,Maine, USA) in 100 mM Tris, 100 mM EDTA, pH 8.0, was added to 300 μl ofbacterial cell suspension. Plugs were immediately poured in sterilemoulds and kept on ice until hardened. The plugs were then incubated at50° C. overnight in sterile 1.5 ml 0.5 M EDTA, 1% N-lauroylsarcosin, 2mg/ml proteinase K (Roche Diagnostics), pH 8.0, by shaking. The nextday, the plugs were thoroughly washed 5 times over the whole day at roomtemperature in sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) andstored in sterile 0.5 M EDTA, pH 8.0, at 4° C. until further use. Todigest the plugs they were first incubated in 4×Buffer H (RocheDiagnostics) for 10 min at 22° C. Then the plugs were incubated at 37°C. by shaking for 7½ hrs in 2× Buffer H containing 40 U of NotI (RocheDiagnostics). They were then placed into the slots of a 1% Sea Kem goldagarose gel in 0.5×TBE and sealed with 1% Sea Kem gold agarose. The gelwas then equilibrated in 0.5×TBE at 12° C. using an ElectrophoresisCHEF-DR® III system (BioRad Laboratories, Hercules, Calif., USA). Toseparate NotI DNA fragments, the field was 6V/cm, having an angle of120°, starting with 1 s and ending with 12 s. The duration of the PFGEwas 14 hrs and it was performed at 12° C. The gel was stained 30 min atroom temperature in water containing 0.5 μg/ml ethidium bromide, washedtwo times with water and analyzed under a UV-light. Additionally, thegel was further used for Southern-blotting.

Southern-Blot Analyses:

Southern-blotting was done by alkaline transfer onto positively chargednylon membranes (Roche Diagnostics) with an LKB 2016 VacuGene vacuumblotting pump (Pharmacia LKB). To depurinate the agarose gels they wereincubated for 10 min in 0.25 M HCl, and subsequent transfer wasperformed with 0.4 M NaOH for 1½ hrs. After blotting, membranes werebaked for 30 min at 80° C. under vacuum. After at least one hr ofprehybridization, hybridization was carried out in 5×SSC (1×SSC in 0.15M NaCl plus 0.015 M sodium citrate)-1% blocking reagent (RocheDiagnostics)-0.1% N-lauroylsarcosine sodium salt-0.02% sodium dodecylsulphate (SDS) at 68° C. overnight, using DIG-labeled DNA as probe.Membranes were washed under nonstringent conditions twice for 5 min eachwith 50 ml of 2×SSC-0.1% SDS per 100 cm² at 22° C., followed bymedium-high-stringency washing twice for 15 min each with 50 ml of0.2×SSC-0.1% SDS per 100 cm² at 68° C. The membranes were then processedwith phosphatase-labeled anti-DIG antibody (Roche Diagnostics) accordingto the manufacturer's protocol. Signals were produced withchemiluminescent substrate (CSPD, Roche Diagnostics).

Pulsed-field gels were treated for Southern-blotting by using the samesolutions as described above. To depurinate the agarose gelsefficiently, they were incubated for 20 min in 0.25 M HCl, and thenequilibrated for 20 min in 0.4 M NaOH. Transfer was performed for 3 hrsand the gels were treated as described above.

Expression and Purification of His-Tailed Fusion Protein AcrV:

Oligonucleotide primers used to amplify the whole acrV gene are given inTable 2. The PCR reactions were carried out as described above with theexception of using Pwo DNA polymerase (Expand Long Template PCR Systemkit, Roche Diagnostics) instead of Taq DNA polymerase and genomic DNA ofA. salmonicida JF2267. The PCR products were purified by using the HighPuree PCR Product Purification Kit (Roche Diagnostics) as described bythe manufacturer's protocol. Then the acrV PCR product was cloned intopGEM-T vector (Promega, Madison, Wis. USA), having 3′-T overhangs at theinsertion sites, as described in the manufacturer's protocol andtransformed into E. coli strains XL-1 Blue. The resulting plasmid wasdesignated pJFFIVB873. The cloning of the PCR products into pGEM-Tvector was used to provide efficient restriction of the subclonedfragments. Plasmid pJFFIVB873 was then digested with EcoRI and NotI, andthe DNA fragment was inserted into the T7-promoter-based expressionvector pETHIS-1 (Schaller et al., 1999). The resulting plasmid,pJFFETHISacrV4 was purified and controlled by DNA sequencing to assurethe fusions with the vector's poly-His codons and then transformed intoEscherichia coli BL21 (DE3) cells (Novagen) for expression. Expressionwas induced by addition of 1 mM IPTG to cultures and incubationcontinued for another 3 h. The cells were sedimented by centrifugationat 3000×g for 10 min, resuspended in 5 ml PN buffer (50 mM NaH₂PO₄, pH8.0, 300 mM NaCl), sonicated with a microtip for 4 min with the poweroutput control at 1 and a duty cycle of 50% (1 s pulses) in a BransonSonifier 250 (Branson Ultrasonics, Danbury, Conn., USA). Then guanidinehydrochloride was added to a final concentration of 6 M and wasincubated overnight at 4° C. on a shaker. The mixture was loaded onto aprewashed 2.5 ml bed volume Ni²⁺ chelation chromatography column(Qiagen) and washed once more with 30 ml PNG buffer (50 mM NaH₂PO₄, pH8.0, 300 mM NaCl, 6 M guanidine hydrochloride). Step elutions of theproteins were performed by adding 10 ml PNG buffer at each different pH(7.0, 6.0, 5.5, 5.0, and 4.5) and fractions of 1 ml were collected. Thefractions were dialyzed and analyzed on 15% PAGE. The purified fusionproteins were eluted at pH 4.5.

Production of Monospecific Rabbit Anti-AcrV Antibodies and ImmunoblotAnalyses:

Monospecific, polyclonal antibodies directed against AcrV were obtainedby immunizing rabbits subcutaneous with 80 μg of recombinantpolyhistidine-tailed AcrV protein in 200 μl PN buffer and 150 μl NaCl(0.85%) mixed with 350 μl Freund's complete adjuvant (DifcoLaboratories, Detroit, Mich., USA) followed by a booster immunizationwith the same amount of protein in Freund's incomplete adjuvant (Difco)3 weeks later. The animals were bled 22 d after the booster immunizationaccording to standard protocols (Harlow and Lane, 1988).

Infection of Fish Cell Cultures with A. salmonicida:

Rainbow trout (Oncorhynchus mykiss) gonad cells (RTG-2, ATCC CCL-55)were grown in 75 cm² tissue culture flasks (Techno plastic products AG,Trasadingen, Switzerland) at 22° C. in minimum essential medium(GibcoBRL Life Technologies, Basel, Switzerland) supplemented with 2 mML-glutamine (GibcoBRL), 1× non-essential amino acids (GibcoBRL), 3 g/lsodium bicarbonate and 10% foetal bovine serum. Three days beforeinfection the cells were trypsinized and 4 mio cells were seeded into a25 cm² tissue culture flask. Monolayered RTG-2 cells were infected withA. salmonicida cells resuspended in phosphate buffered saline (PBS) pH7.4 at a multiplicity of infection of 20:1 or 2:1 (bacteria/fish cells).As a control also 100 μl of pure PBS pH 7.4 were added to cultured fishcells. After 24 hrs of infection at 15° C. the fish cells werephotographed under a green filtered phase contrast microscope (Aixovert100, Zeiss, Jena, Germany). To detach the cultured cells from the flask,the flask was shaken by hand. The suspended cells were centrifuged for 5min at 4,000 rpm. Lysis of the fish cells was performed in 100 μldistilled water with two subsequent freeze thawing steps and verified bymicroscopy. The lysed fish cells were used for further analyzes onWestern-blots.

Protection Assay Using Rabbit Antiserum AcrV:

RTG-2 fish cells were grown as described above. Two days beforeinfection 20 mio of trypsinized RTG-2 fish cells were seeded into 24well culture plates (1.9 cm²) (Techno plastic products AG, Trasadingen,Switzerland). Rabbit antiserum directed against AcrV as well as controlpreserum were decomplemented for 30 min at 56° C. A fresh culture of A.salmonicida (at end exponential growth phase) was washed and resuspendedin PBS pH 7.4 and mixed with either preserum or anti AcrV antiserum at aratio of 1:1, 1:10, 1:100, 1:1000 or 1:10,000. Bacteria were incubatedwith the serum at 18° C. for 30 min. The opsonized bacteria were addedto the fish cells in a ratio of 20:1 or 2:1 (bacteria/fish cells). After21 hrs of infection at 15° C. the fish cells were photographed asdescribed before and inspected for morphological changes.

SDS-PAGE and Immunoblot Analysis:

Proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE)as described by Laemmli (Laemmli, 1970) using 15% or 10% polyacrid gelsand transferred to a nitrocellulose membrane (BioRad Laboratories). Forimmunoblotting, Western-blots were blocked with 1% milk buffer for atleast one hour and then incubated with the rabbit antiserum AcrV(1:2000) or with the rabbit preserum (1:1000) in milk buffer overnightat 4° C. The membranes were then washed thoroughly with water beforephosphatase-labelled conjugate (Goat anti-Rabbit IgG (H+L) [cat. no.075-1506], Kirkegaard & Perry, Gaithersburg, Md., USA) diluted 1:2000 inmilk buffer was added. The reaction was visualized 90 min later byincubation with BCIP-NBT (Ausubel et al., 1999).

EXAMPLES/RESULTS

Cloning and Sequence Analysis of the virA Locus of a Type III Pathway ofA. salmonicida:

Analysis of A. salmonicida strain JF2267 with an array of broad rangeprobes for detection of type III secretion pathways revealed a strongsignal with the lcrD subset of the probes, indicating the presence of anew type III secretion pathway. Subsequent Southern-blot analyses showeda 4.8 kb fragment of SacI-SalI digested genomic DNA of strain JF2267reacting with the lcrD probe. This fragment was cloned on vectorpBluescriptII-SK⁻ leading to plasmid pJFFIVB638 which was subsequentlysequenced. DNA sequence analyses revealed the presence of eight openreading frames (ORF) (FIG. 1) which showed strong similarity to thegenes encoded on the virA operon of the type III secretion pathway ofYersinia pests and Pseudomonas aeruginosa. In analogy to the Y. pestisgenes, we named them acr1, acr2, acr3, acr4, and acrD (Aeromonas calciumresponse (FIG. 1)). They are located on a single operon followed by atranscription termination signal similar to the virA operon of Y.pestis, Y. enterocolitica and Pseudomonas aeruginosa (Boland et al.,1996; Iriarte and Cornelis, 1999; Plano et al., 1991; Cornelis, 1998;Yahr et al., 1997a). The similarities of the genes acr1, acr2, acr3,acr4 and acrD with the analogues in Y. enteroclitica and in P.aeruginosa are given in Table 2. Downstream lcrD we identified a locuswith a canonical promoter sequence followed by further genes named acrR,acrG, and acrV on a separate operon (FIG. 1) according to thecorresponding genes in Y. pestis (Table 3) (Barve and Straley, 1990;Skrzypek and Straley, 1993; Nilles et al., 1998). The ORF of theputative acrV gene seemed to be incomplete on the 4.8 kb SacI-SalIfragment of pJFFIVB638, and represented only the 5′-half of the gene.The remaining part of acrV and part of acrH located downstream of acrVwere cloned separately from the λ phage gene library of A. salmonicidaas an overlapping clone which was obtained by screening the gene libraryusing a gene probe for the 5′-half of acrV which was produced by PCRwith primers AcrV-L and AcrV-R (Table 2). The resulting plasmid based onvector pBK-CMV was designated pJFFIVB832. From this plasmid, a 0.9 kbSalI fragment containing the 3′ end of acrV and part of the downstreamgene acrH was subcloned on pBluescriptII-SK and designated pJFFIVB828.

Instability of the Genes Belonging to the Type III Pathway in A.salmonicida:

When we analyzed the different A. salmonicida strains with a specificprobe for acrD, we discovered by using Southern blot hybridization thatthe acrD gene was present only in strain JF2267 but not in thederivative strain JF2397 which had undergone nine passages of subsequentsingle colony cloning isolation. Additionally, the type strain of A.salmonicida, ATCC 33658^(T), did not show a signal with the acrD probe.However, several A. salmonicida strains that were freshly isolated fromsalmon and trout with furunculoses did contain acrD (Table 4). Theseresults indicate that the type III secretion pathway of A. salmonicidamay be lost easily. In order to get an estimate on the loss of the typeIII secretion genes, we have analyzed the kinetics of disappearance ofacrD after a shift of growth temperature of strain JF2267 from 19° C. to22° C. Colony hybridization with the acrV probe revealed that in a freshculture of strain JF2267, the acrD gene was present in all cells grownat 19° C. After the shift to 22° C., acrD was still present for further5½ hrs, following which it was lost very rapidly within less than 1 hr(FIG. 2). Taking into account the generation time of 2 h for A.salmonicida under the given growth conditions, the acrD gene was lostwithin two generations. To analyze the loss of acrD further, undigestedand NotI digested genomic DNA of A. salmonicida strain JF2267 and of theacrD deficient derivative strain JF2397 were submitted to pulse fieldgel electrophoresis (PFGE) and subsequent Southern blot hybridizationwith the acrD probe. PFGE analyses of total undigested DNA revealed thepresence of two large plasmids in strain JF2267 while in strain JF2397only one of the two plasmids was seen (FIG. 3). Digestion of the totalDNA from these two strains with the rarely cutting enzyme NotI revealedthe lack of a 84 kb band in strain JF2397 compared to JF2267 as the soledetectable difference (FIG. 3). Southern-blot hybridization of the DNAon this gels with the acrD probe confirmed the larger plasmid and the 84kb NotI fragment of strain JF 2267 to contain acrD gene. Neither theremaining large plasmid in JF2397 nor any of its NotI fragmentshybridized with the AcrV probe. This indicates that the type IIIsecretion genes, or at least the virA operon thereof, are located on alarge plasmid in the size range of 84 kb.

Presence of acrD in A. salmonicida Strains:

In order to assess the presence of the acrD gene in various A.salmonicida strains, DNA samples extracted from A. salmonicida Typestrain ATCC33658 and various field strains isolated from salmon or charwere digested with restriction enzymes SalI and SacI, separated by 0.7%agarose gel electrophoresis, blotted onto nylon membranes and hybridizedwith the acrD gene probe. The Southern blot revealed the presense of theacrD gene on a 4.8 kb fragment in all strains except in the type strainATCC33658, the laboratory strain JF2396 which was used for the type IIIsecretion genes, and A. salmonicida strain MT44 known to be a virulentfor trout. One field strain, # 24, showed a very weak hybridizationsignal indicating that the culture contains acrD only in a minorpopulation of the cells (Table 1).

Infection of RTG-2 Fish Cells and Protection of Cell Damage withAnti-AcrV Antiserum:

Freshly cultured A. salmonicida strain JF2267 was used to infect RTG-2cells. After 24 hrs of incubation the fish cells were rounded up andalso detached from the plastic support (FIG. 4A). In contrast cellsinfected with A. salmonicida type strain ATCC 33658^(T) or strain JF2397(FIGS. 4B and C), both known to be devoid of acrD and acrV, showed nomorphological changes at all in spite of a massive multiplication of thebacteria in the cultures. RTG-2 fish cells which were incubated with PBSbuffer as control showed no morphological changes like the cellsinfected with the acrD and acrV deficient strains JF2397 or ATCC33658^(T) (FIG. 4D).

In order to study further the role of the newly detected type IIIsecretion pathway in virulence of A. salmonicida, we incubated strain JF2267 with monospecific polyclonal anti-AcrV antibodies prior toinfection of RTG-2 fish cell cultures. When RTG-2 fish cells wereinfected with strain JF2267 that was incubated with rabbit anti-AcrVantibodies diluted 1:1 or 1:10, the characteristic morphological changesof the cells were reduced, significantly affecting only 20% of the cellsor less (FIG. 4E) compared to the infection with non-treated strain JF2267 (FIG. 4A) or to the infection with JF 2267 that was pretreated withserum from the same rabbit taken before immunization (FIG. 4F).Titration of the anti-AcrV serum showed that protection of about 50% ofthe RTG-2 cells could still be reached with a serum dilution of 1:100,while further dilutions had no visible effect in protection.

Expression of AcrV in A. salmonicida:

The expression of AcrV in A. salmonicida strain JF2267 was assessed byimmunoblots using AcrV-His antibodies. When A. salmonicida was grownunder standard culture conditions in TSB medium, no AcrV protein couldbe detected from total cells nor from culture supernatant of strain JF2267, nor in the control of strains JF2397 and ATCC33658^(T). However,when the cells are submitted to a low Ca²⁺ response by chelating freeCa²⁺ ions in the growth medium by the addition of 10 mM NTA, we detectedAcrV with anti-AcrV antibodies in the pellet of JF2267 as a protein ofabout 37 kDa (FIG. 5) but not in strains JF2397 and ATCC33658^(T), whichare both devoid of the AcrV gene (FIG. 5). No AcrV protein could bedetected in the supernatants of cultures from strains JF2267, JF2396 andATCC33658^(T), grown in Ca²⁺ depleted medium.

When strain JF2267 was grown under standard culture conditions(containing free Ca²⁺ ions) and then put in contact with RTG-2 cells ata ratio 2:1 (bacteria: cells) for 30 minutes, the AcrV protein could bemonitored on immunoblots reacting with anti-AcrV, similar to culturesfrom Ca²⁺ depleted medium.

Recombinant AcrV Vaccine Trial

(see Appendix A)

While particular elements, embodiments and applications of the presentinvention have been shown and described, it will be understood, ofcourse, that the invention is not limited thereto, since modificationsmay be made by those skilled in the applicable technologies,particularly in light of the foregoing description. The appended claimsinclude within their ambit such modifications and variants of theexemplary embodiments of the invention described herein as would beapparent to those skilled in the applicable technologies

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Appendix A

Recombinant AcrV Vaccine Trial

Materials:

Vaccine Formulations:

-   -   1. The AcrV vaccine was formulated using recombinant,        Histidine-tagged AcrV resuspended in 10 mM phosphate buffer, pH        7.0, to 112.5 μg/mL. Four parts of this protein solution were        mixed with one part oil adjuvant for a final AcrV concentration        of 90 μg/mL. The dose for testing was 0.1 mL, or 9 μg/fish.    -   2. The commercial comparator vaccine was serial 4-13 of the        vaccine MultiVacc4 (Bayotek International Ltd.)    -   3. The placebo (control) vaccine consisted of phosphate buffered        saline (PBS) (10 mM phosphate, 150 mM NaCl, pH 7.2).    -   4. All vaccines were maintained at 4° C. until use.        Methods        Trial Design:

Fish (rainbow trout Oncorhynchus mykiss) that have been determined to bepathogen free and are at least 15 g in size are held for at leastone-week pre vaccination for acclimation purposes. During theacclimation period the fish are offered 1% body weight in salmonid fishfood every day, however they are denied food 24 hours pre andpost-vaccination.

At least 50 fish are vaccinated 0.1 mL of AcrV vaccine viaintra-peritoneal (IP) injection, or 0.2 mL of the commercial vaccineMultiVacc4. At the same time a group of at least 50 fish from the samestock are mock vaccinated with 0.1 mL of PBS. Vaccinated fish are thenheld for a period of at least 350-degree days to allow specific immuneresponse generation in an acclimation tank with a continuous flow ofwater at a temperature of 12-13° C. The fish are offered 1% body weightin salmonid fish food daily until 24 hours pre-challenge andpost-challenge.

After at least 350-degree days post vaccination 50 fish per group werechallenged by IP injection with a pre-determined concentration ofvirulent Aeromonas salmonicida. The dosage depends on the source of thefish and the water temperature (this is determined empiricallyimmediately prior to challenge of test fish). The identical procedure isperformed with the placebo vaccinated control fish. The fish areobserved daily for mortality for 21 days post challenge and the cause ofmortality assessed and examined to ensure that mortality is attributedto the challenge organism. After 24 hours post-challenge the fish areagain offered 1% body weight in salmonid fish feed daily. Tanks aremaintained with a continuous flow of water at a temperature of 12-13° C.For a challenge series to be considered satisfactory; all challengegroups must meet the following criteria:

-   -   1. At least 70% of the non-immunized controls must die within 21        days of challenge.    -   2. A relative percent survival (RPS) of no less than 25% must be        achieved for the challenge disease before a vaccine is        considered even partially efficacious for this disease.        RPS=[1-(% mortality vaccinates/% mortality controls)]×100        Developed from: The Rules Governing Medicinal Products in the        European Union, Volume VII, Guidelines for the testing of        veterinary medicinal products. 1994. Specific Requirements for        the Production and Control of Live and Inactivated Vaccines        Intended for Fish. Section 3.2. Potency.

Results Group % Mortality RPS PBS 82 — AcrV 49 40 MultiVacc4 30 63

1. There was a strong challenge with 82% control mortalities. TABLE 1 A.salmonicida strains used in this study and presence of acrD strainorigin acrD^(a)) ATCC33658 American Type Culture Collection, Type strain− JF2267 Char (Savelinus alpinus), Switzerland + JF2396 Laboratorystrain, derivative of JF2267 − CC-23 Salmon, Norway + CC-24 Salmon,Norway +/−^(b)) CC-27 Salmon, Norway + CC-29 Salmon, Scotland, UK +CC-30 Salmon, Canada + CC-34 Salmon, Canada + MT 44 Spontaneous nonvirulent mutant − CC-63 Salmon, Canada + CC-72 Salmon, Canada +^(a))as determined by Southern blot hybridization^(b))very weak hybridization signal indicating that only a minor part ofthe population of the culture contains the acrD gene

TABLE 2 Oligonucleotide primers Annealing Name Seguence^(a) 5′ to 3′Position^(b) temp. ° C. AslcrD-L^(c) GCCCGTTTTGCCTATCAA 1159-1176 60AslcrD-R^(c) GCGCCGATATCGGTACCC 2028-2011 60 AcrV-L^(c)TTCGTCGGCTGGCTTGATGT 4144-4163 58 AcrV-R^(c) GAACTCGCCCCCTTCCATAA4734-4715 58 AsacrVt-L^(d) gggaattcGATGAGCACAATCCCTGACTAC 4104-4125 57AsacrVt-Rd atgcggccgcAAATTGCGCCAAGAATGTCG 5188-5169 57 AsacrVN′-R^(d)tcgcggccgcACCCTTTACGCTGATTGTC 4555-4537 57 AsacrVC′-L^(d)cggaattcGTTGCGGGATGAGCTGGCAG 4554-4573 57 AsacrVC′-R^(d)tcgcggccgcACTCGGCTTCTATGCCACTC 4987-4968 57^(a)Lowercase letters indicate nucleotides added to create restrictionenzyme recognition sites (underlined) for cloning.^(b)Based on nucleotide sequence of A. salmonicida JF2267^(c)Primer used for gene probe preparation^(d)Primer used for amplification of gene acrV, acrV-N, and acrV-Crespectively

TABLE 3 A. salmonicida type III proteins compared to analogues In P.aeruginosa and in V. entercolitica. Protein in Analogue in Similarlty/Genbank Analogue in Similarity/ Genbank A. salmonicida P. aeruginosaidentity^(a)) access. nr. Y. enterocolitica Identity^(a)) access. nr.Proposed function Acr1 Pcr1 80/60 AF019150 TyeA 83/69 AF102990 part ofthe translocation-control apparatus, required for selectivetranslocation of Yops Acr2 Pcr2 63/44 AF019150 SycN 77/62 AF102990chaperone forYopN Acr3 Pcr3 62/47 AF019150 YscX 69/54 AF102990 part ofthe type III secretion apparatus, secretion of Yop Acr4 Pcr4 66/55AF019150 YscY 64/52 AP102990 part of the type III secretion apparatus,secretion of Yop AcrD PcrD 90/82 AF019150 LcrD 90/82 X87771 innermembrane spanning protein of type III secretion AcrR PcrR 68/58 AF019150LcrR 71/58 AF102990 AcrG PcrG 63/46 AF010149 LcrG 64/42 AF102990regulation of low calcium response AcrV PcrV 50/35 AF010149 LcrV 53/37X96797 regulation of low calcium response, sensor suppression of TNFáand Interferon ã, protective antigen AcrH PcrH 78/65 AF010149 LcrH(SycD) 79/58 AF102990 regulation ot low calcium response, chaperon forYopD, secretion^(a))given as % of similar/identical amino acids

1-22. (canceled)
 23. An isolated polypeptide comprising at least oneepitope or epitopic region of a selected one of the class Acr1; Acr2;Acr3; Acr4; AcrD; AcrR; AcrG; AcrV; and AcrH.
 24. An isolated nucleicacid fragment encoding a protein having an amino acid sequence as givenin a selected one of the class comprising SEQ ID NO:1; SEQ ID NO:2; SEQID NO:3; SEQ ID NO:4; SEQ ID NO: 5; SEQ ID NO:6; SEQ ID NO: 7; SEQ IDNO:8; and SEQ ID NO:9.
 25. An isolated nucleic acid fragment comprisingSEQ ID NO:10, or the complement thereof.
 26. An immunogenic,immunological or vaccine composition comprising a polypeptide as claimedin claim
 23. 27. An immunogenic, immunological or vaccine compositioncomprising a nucleic acid fragment of claim
 24. 28. An immunogenic,immunological or vaccine composition comprising a nucleic acid fragmentof claim
 25. 29. A method for reducing the susceptibility of fish toinfection by a virulent strain of A. salmonicida comprising theintraperitoneal, intramuscular, intradermal, intracellular, spray,immersion, or oral administration to said fish of a compositioncomprising an immunogenic amount of at least one epitope or epitopicregion of AcrV, any other protein of the A. salmonicida Type IIIsecretion apparatus, a natural or genetically modified variant thereof,or an antigenic peptide derived or synthesized thereof.
 30. The methodof claim 29, wherein the at least one epitope or epitopic region of aprotein of the A. salmonicida Type III secretion apparatus, a natural orgenetically modified variant thereof, or an antigenic peptide derived orsynthesized thereof, is fused to at least one other polypeptide at theN′-terminal, C′terminal, or both, and wherein the said at least oneother polypeptide facilitates expression.
 31. The method of claim 29,wherein the at least one epitope or epitopic region of a protein of theA. salmonicida Type III secretion apparatus, a natural or geneticallymodified variant thereof, or an antigenic peptide derived or synthesizedthereof, is fused to at least one other polypeptide at the N′-terminal,C′-terminal, or both, and wherein the said at least one otherpolypeptide facilitates the formation of insoluble intracellularaggregates.
 32. The method of claim 29, wherein the at least one epitopeor epitopic region of a protein of the A. salmonicida Type III secretionapparatus, a natural or genetically modified variant thereof, or anantigenic peptide derived or synthesized thereof, is fused to at leastone other polypeptide at the N′-terminal, C′-terminal, or both, andwherein the said at least one other polypeptide is a T cell epitope or aB cell epitope.
 33. A method for reducing the susceptibility of fish toinfection by a virulent strain of A. salmonicida comprising theintraperitoneal, intramuscular, intradermal, intracellular, spray,immersion, or oral administration to said fish of an immunogenic amountof a composition comprising the acrV gene, the gene of any other proteinof the A. salmonicida Type III secretion apparatus, homologues,fragments, or synthetic oligonucleotides derived thereof.
 34. A methodfor reducing the susceptibility of fish to infection by a virulentstrain of A. salmonicida comprising the intraperitoneal, intramuscular,intradermal, intracellular, spray, immersion, or oral administration tosaid fish of an immunogenic amount of a composition comprising theisolated nucleic acid fragment of SEQ ID NO:10.
 35. A therapeutic methodfor the protection of fish from the toxic effect of a virulent strain ofA. salmonicida comprising the use of antiserum directed against AcrV,variants or fragments thereof, or synthesized peptides thereof.
 36. Themethod of claim 35 wherein the antiserum is directed against recombinantAcrV.